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Journal of the Chilean Chemical Society

versión On-line ISSN 0717-9707

J. Chil. Chem. Soc. v.49 n.2 Concepción jun. 2004

http://dx.doi.org/10.4067/S0717-97072004000200006 

 

J. Chil. Chem. Soc., 49, N 2 (2004), pags.:137-141

SECONDARY METABOLITES IN THE EPICUTICLE OF HAPLOPAPPUS FOLIOSUS DC. (ASTERACEAE)

 

ALEJANDRO URZÚA

Faculty of Chemistry and Biology, University of Santiago of Chile, Santiago, Chile E-Mail : urzuamoll44@yahoo.com


SUMMARY

From the CH2Cl2 extract of the surface of Haplopappus foliosus, monoterpenes, sesquiterpenes, flavonoids, coumarins, phenyl propanoids, n-alkanes and different miscellaneous compounds were identified. The complexity of the chemistry in terms of number of families and compounds makes attractive the use of this specie as a model to study variations in the cuticle chemistry, triggered by different ecological pressures.

Keywords: Haplopappus foliosus; Monoterpenes; Sesquiterpenes; Flavonoids; Coumarins; phenyl propanoids, n-Alkanes; miscellaneous compounds.


INTRODUCTION

Haplopappus foliosus DC. is an herbaceous shrub, widely distributed in the coast of the Chilean IV and V Regions ( 32° S to 34° S )1). Some populations of this species grow in sandy soils near to the sea coast, where high levels of humidity are found. Other populations grow several km from the coast, on sunny and degraded arid soils, under very dry conditions 1). The extreme differences of ecological conditions to which this species is exposed, has led to the development of populations with very different morphological characteristics. This is also reflected in the cuticle chemistry, that shows important differences between groups of plants that shares different ecological environments (A. Urzúa unpublished results).

The cuticle constituents of H. foliosus have been identified. They include nine monoterpenes, two sesquiterpenes, n-alkanes, (E)-cinnamic acid, (E)-cinnamic acid benzyl esters and some miscellaneous compounds2). Also, a recent communication reports the isolation of two new antibacterial clerodane diterpenoids3).

It was apparent in those communications that the surface chemistry of H. foliosus is extremely complex and that most of the compounds present in the cuticle are not yet identified. In order to have a more complete view of the cuticle composition an exaustive study of the surface chemistry of H. foliosus, was undertaken. A total of 85 compounds were identified , including: monoterpenes; sesquiterpenes; flavonoids; coumarins; n-alkanes; n-alkenes, phenyl propanoids and miscellaneous compounds.

This result supports the use of Haplopappus foliosus as a model plant to study variations of the surface chemistry triggered by different ecological pressures. In addition H.foliosus is the host plant of of Trupanea Schrank ( Diptera: Tephritidae) 4) species. A thorough understanding of the external chemistry of H.foliosus may shed light on novel aspects of the interaction between this plant and its insects hosts.

EXPERIMENTAL

Plant material

A representative sample of aerial parts of Haplopappus foliosus DC. (Asteraceae) was collected during the flowering season, October 1999, from a population found near the coast of Zapallar (V Region, Chile 32° 30'S, 71° 30'W). Voucher specimens (SGO-111557) were deposited in the Herbarium of the National History Museum, Santiago, Chile.

Plant extraction

The external compounds of a representative sample of Haplopappus foliosus DC. (leaves and stems, 500 g) were extracted, in the field, by immersion of the fresh plant material in CH2Cl2 (5.0 L) for 15-20 s. The procedure was repeated twice.

Column chromatography separation of the CH2Cl2 extract

The CH2Cl2 extract (8.65 g, 1.73 %) was fractioned by CC (silica gel) using pentane-CH2Cl2 and CH2Cl2-MeOH step gradients to afford eleven fractions.

TLC study of the extracts and fractions

TLC of the extract and fractions was performed on silica gel 60 F254 pre-coated plates from Merck. Specific spray reagents were used for detection of different families of compounds5): anisaldehyde-H2SO4 , phosphomolibdic acid and vanillin-H3PO4 for terpenoids, diphenylboric acid-b-ethylamino ester-4000(PEG) for flavonoids. Coumarins were detected without chemical treatment as fluorescent zones at UV-366 nm, after developing the plates with toluene-ether (1:1) saturated with 10% acetic acid.

GC-EM study of the extract and fractions

The CH2Cl2 extract and the fractions obtained by CC were analyzed in triplicate, by GC-MS using a FISONS MD-800, equipped with a HP DB-5-MS capillary column (15 m x 0.20 mm). The temperature of the injector was 285 °C, and the programmed temperature of the column started at 40 °C, for 2 min, followed by a rise to 310 °C at 8 °C min-1. The column was then kept isothermally for 5 min. Helium was the carrier gas at 7 psi. Detection was done by EI and QI. The identification of the compounds was achieved by comparison of the retention times with standards, and the mass spectra were compared with data from the NIST library only when the correlation index was greater than 98%2). The percentage of some families of compounds was calculated from the peak areas of the chromatograms.

Derivatization of fractions 8, 9 and 11 was done treating the sample with 200 mL of N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) (Aldrich), to obtain the TMS derivatives.

Nomenclature of compounds

Names of monoterpenoids, diterpenoids and flavonoids are given according to the Handbook of Terpenoids6) and Flavonoids advances in research since 1986 7).

RESULTS AND DISCUSSION

Chemical composition

The CH2Cl2 extract (8.65 g, 1.73 %) was fractioned by CC (silica gel) using pentane-CH2Cl2 and CH2Cl2-MeOH step gradients to afford eleven fractions: F1 (0.70g), F2 (0.60g), F3 (0.60g), F4 (0.60 g), F5 (0.60g), F6 (0.60g), F7 (1.1g), F8 (1.2g), F9 (0.70g), F10 (0.40g) and F11 ( 1.25g).

The compounds identified in the fractions obtained from the CC separation of the CH2Cl2 extract were: Monoterpenoids: p-cymene (1) , a-terpinene (2), limonene (3), g-terpinene (4) , p-menth-1-en-4-ol (5) , origanol (6) , trans-p-menth-2-en-1-ol (7), cis-p-menth-2-en-1-ol (8), (Z)-a-ocimene (9), 3-carene (10), thujane (11), pinocarveol (12), b-pinene (13), borneol (14), 5-(acetyloxy)-4,6,6-trimethyl-endo-biciclo [2.2.1]heptan-2-one (15), 5-hydroxy-4,6,6-trimethyl-endo-biciclo [2.2.1]heptane (16), tricyclene (17) and 1,5-dimethyl-6-methylenespiro[2.4]heptane (18).

Scheme 1

Sesquiterpenoids: 4(15), 7(11)-eudesmadiene (19), 3,11-eudesmadiene (20), 5-eudesmen-11-ol (21) , 4-eudesmen-11-ol (22) , cadalene (23) , 4,9-canadiene (24), 3,9-canadiene (25), g-canadiene (26), 1a-cadin-4-en-10-ol (27) ,1(10),11-eremophiladiene (28) , 1(5)-guaien-11-ol (29) , decahydro-4,8a-dimethyl-7-(1-methylethenyl)azulene (30) , copaene (31) , a-bisabolol (32), 10(14)-aromadendrene (33), a-ionene (34), d-ambrinol (35) and 5-(1,1-dimethylethenyl)-2,3-dihydro-3, 3-dimethyl-1H-inden (36).

Scheme 2


Phenylpropanoids: eugenol (37), a-asarone (38) , elemicin (5-allyl-1,3,3-trimethoxybencene) (39), (Z)-cinnamic acid (40), (E)-cinnamic acid (41) , pentyl-(E)-cinnamate (42) , benzyl-(E)-cinnamate (43), (2-phenylethyl)-(E)-cinnamate (44) and iso-butyl-(E)-cinnamate (45).

Scheme 3

Coumarins: aesculetin (6,7-dihydroxycoumarin) (46), scopoletin (6-methoxy-7-hydroxycoumarin) (47), scoparone (6,7-dimethoxycoumarin) (48) and prenyletin (49).

Scheme 4

Flavonoids: kaempferol (3,5,7,4¢-tetrahydroxyflavone) (50), isokaempferide (5,7,4¢-trihydroxy-3-methoxyflavone) (51), ermanin (5,7-dihydroxy-3,4¢-dimethoxyflavone) (52), kumatakenin (5,4¢- dihydroxy-3,7-dimethoxyflavone) (53), isorhamnetin (3,5,7,4¢-tetrahydroxy-3¢-methoxyflavone) (54),rhamnazin (3,5,4¢-trihydroxy-7,3¢-dimethoxyflavone) (55) and eupatolitin (3,5,3¢,4¢-tetrahydroxy-6,7-dimethoxyflavone (56).

Scheme 5

Miscellaneous Compounds: 4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-2-butanone (57), tetrahydroactinidiolide (58), 4,4-dimethyl-2-allylcyclohexanone (59), 3,4-dihydro-a-ionone (60), benzaldehyde (61) , p-vinylbenzaldehyde (62), ethyl resorsinol (63), isopropylphenol (64), m-hydroxyacetophenone (65), 2,3 dihydrobenzofurane (66), 2,4-dimethyl-3-oxapentane (67), 2,3-dichloro-2-methylpropanal (68), 4-phenyl-2-azetidinone (69) and 1,2,3,4,5,6,7,8-octahydro-1-methylphenantrene (70).

Scheme 6

n-Alkanes: dodecane (C12H26), tetradecane ( C14H30 ), hexadecane (C16H34) ,octadecane (C18H38), tricosane (C23H48) , pentacosane (C25H52), hexacosane (C26H54) ,heptacosane (C27H56), octacosane (C28H58), nonacosane (C29H60) , triacontane (C30H62) , untriacontane (C31H64 ) , dotriacontane (C32H66), tritriacontane (C33H68). n-Alkenes: 11-tricosene (C23H46).

Identification of the components

Fractions 1 and 2, eluted with pentane were analyzed by GC-EM-NIST library and showed the presence of non oxygenated mono and sesquiterpenoids: 1-4, 9-11, 13, 17,18-20, 23-26, 28, 30-34 and 36. The fractions also showed the following miscellaneous compounds: 59, 62 and 68-70. Identification of compounds 1-4, 9-11, 13, 23, 32 and 33 was also confirmed by direct comparison (CG-EM) with authentic samples.

The n-alkanes were found exclusively in F1 and F2. Using the software that select compounds with m/z 57,71,85 and 99 and triacontane as standard, the total amount of n-alkanes was estimated as 8% of the total external compounds. Tricosane, pentacosane ,nonacosane and untriacontane constituted around 80% of the alkane fraction.

Fraction 5 and 6 eluted with CH2Cl2 were analyzed by GC-EM-NIST library and showed the presence of the phenylpropanoids: 37-45. Identification of compounds 37 and 40-45 was also confirmed by direct comparison (CG-EM) with authentic samples.

Fraction 7 and 8 eluted with CH2Cl2 were analyzed by GC-EM-NIST library and showed the presence of miscellaneous compounds: 60,61,63-66. Identification of compounds 61 and 63-66 was also confirmed by direct comparison (CG-EM) with authentic samples.

Fraction 9 and 10, eluted with CH2Cl2-MeOH, were analyzed by GC-EM-NIST library and showed the presence of the oxygenated mono and sesquiterpenoids: 5-8, 12, 14-16, 21, 22, 27, 29, 32 and 35.

The fractions also showed the oxygenated miscellaneous compounds: 57-58 and 67. Identification of compounds 12, 14, and 67 was also confirmed by direct comparison (CG-EM) with authentic samples.

Fractions 10 and 11, eluted with CH2Cl2-MeOH, showed the presence of flavonoids (TLC) 5). Combination of semi preparative HPLC and TLC yield small amounts of compounds 50 to 56. Compounds 51, 53 and 54 were identified by direct comparison with authentic samples isolated from Haplopappus velutinus Remy8) and Heliotopium spp.9). Compounds 50, 52, 55 and 56 were identified by comparison of its spectroscopic properties with published data7, 12-14).

Fractions 8 and 9, eluted with CH2Cl2, showed the presence of coumarins (TLC and GC-MS).

Preparative TLC yielded small amounts of compounds 46 to 49. The compounds were identified by direct comparison with authentic samples, obtained from Aldrich (Compounds 46- 48) and compound 49 with a sample of prenyletin isolated from Haplopappus multifolius Phil.13). Only the identification of compounds 46 to 48 was also obtained by GC-MS-NITS library. Prenyletin (49) decomposed in the column even after derivatization with MSTFA.

Diterpenoids were detected in F11 as not well defined broad peaks of M + 304, 302, 298, etc. It was apparent that these compounds decomposed in the column. After derivatization with MSTFA nine peaks that correspond to the TMS derivatives of diterpenoids were observed. Two of them were identified as the previously isolated 3,8(17),13-clerodatrien-2-ol-15 oic acid and its acetyl derivative.

The compound 2,3-dichloro-2-methylpropanal (68) is not a contaminant of the solvent, because dichloromethane PRA grade (Aldrich) was used. Is possible to hypothesize that is a metabolite of some pesticide, an OVC (organic volatile contaminant), or a real natural product.

Critical assessment

Haplopappus is well represented in Chile in between 85 1) to 63 14) species, depending on the author. With few exceptions all of them show a characteristic production of external resinous exudates in twigs and leaves. Their biosynthesis occurs in specialized glands populating the surface of all aerial structures of the plants. In addition other specialized structures produce the waxy coating and it is not possible to discard the presence of additional specialized secretory structures.

Although the medicinal properties of the species of Haplopappus are associated with their external chemistry, the first papers on the surface chemistry of one of those species was on H. velutinus 8,15). Using a selective method of extraction of the external compounds, from fresh plant material, labdane diterpenoids and small amounts of flavonoids were isolated. In a second communication the antimicrobial properties and preliminary chemical information of the resinous exudates from twigs and leaves of nine Haplopappus species were informed. The results showed that these species share similar antimicrobial activities although they differ dramatically in the chemical composition 16).

In later communications the chemistry of the resinous exudates of H. diplopappus Remy 17), H. shumanni (O.K.) Br. Et Clark 18), H. deserticola Phil.19) and H. uncinatus Phil.20) was informed. The isolated compounds included labdane and clerodane diterpenoids and also small amounts of flavonoids from H. deserticola and H. uncinatus. It is clear from these works that the compounds isolated from the surface of the Haplopappus species only represent a small proportion of the total fraction. As a matter of fact, with only partial information on the external compounds, studies of plant phenotypic plasticity and ecotypes due to abiotic and biotic factors should be regarded with condition, and may even lead to wrong results 21). In addition, the medicinal properties of the species may be not reproducible due to changes in the concentration of some compounds.

In recent communications, classical isolation and identification of the surface compounds of Haplopappus has been combined with GC-EM spectrometry and HPLC-UV. This new approach has allowed the identification of several new compounds in H. uncinatus, H. foliosus, H.velutinus, H .shumanii and H .illinitus.2, 22) leading to a more complete knowledge of the surface chemistry of species of Haplopappus.

By caring out an exhaustive study of the chemical composition of Haplopappus foliosus, the present work provides the basis for future more accurate investigation of the cuticle composition of this plant under different conditions. These results may thus be regarded as the ground upon which to build a model for the study of the relationship between a plant surface chemistry and the biotic and abiotic factors of its environment.

 

Acknowledgements

This work was supported by FONDECYT (Chile) N 1990209 and by DICYT (Universidad de Santiago de Chile).

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(Received: September 1, 2003 ­ Accepted: December 16, 2003)